C57BL/6 mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) develop acute and chronic demyelinating encephalomyelitis and thereby serve as a useful model for the human disease multiple sclerosis. Demyelination is largely immune-mediated and reflects a balance between pro-inflammatory factors necessary for virus clearance and anti-inflammatory factors critical for limiting bystander tissue damage. Virus clearance requires robust virus-specific CD8 and CD4 T cell responses. In the previous funding period, we showed that optimization of the CD8 T cell response, by mutating a subdominant CD8 T cell epitope, enhanced virus clearance. However, disease course was not changed. We showed that this was due to a pathogenic CD4 T cell response directed at the immunodominant M133 epitope. Only naive M133-specific CD4 T cells were pathogenic because we also showed that memory virus-specific CD4 T cells were protective. We also identified a potent regulatory T cell (Treg, Foxp3+) response directed against the M133 epitope, adding another layer of complexity to the balance between pro and anti-inflammatory factors. To probe these interactions in more detail, we developed mice that were retrogenic or transgenic for expression of an M133-specific T cell receptor. The central objective of this proposal is to investigate in more detail the balance of these immune factors, as delineated in the following specific aims. In Aim 1, factors in M133-specific CD4 T cells that result in pathogenicity (naive T cells) or protection (memory T cells) will be investigated, using our newly developed M133-specific retrogenic and transgenic TCR mice. Adoptive transfer experiments and targeted and genome-wide gene expression approaches will be used in this aim. In Aim 2, the role of M133-specific Tregs in pathogenesis in JHMV-infected mice will be investigated, also taking advantage of the M133- specific TCR transgenic mice. As part of this aim, we will assess whether virus-specific Tregs are more suppressive than bulk Treg populations and whether Tregs enter the memory T cell pool. We will also determine whether pathogen-specific Tregs require expression of a second TCR chain, recognizing a self epitope, for suppressive function.